Myeloglycan, a Series of E-Selectin-Binding Polylactosaminolipids Found in Normal Human Leukocytes and Myelocytic Leukemia HL60 Cells

Glycans containing the GalNAc␤1-4GlcNAc (LacdiNAc or LDN) motif are expressed by many invertebrates, but this motif also occurs in vertebrates and is found on several mammalian glycoprotein hormones. This motif contrasts with the more commonly occurring Gal␤1-4GlcNAc (LacNAc or LN) motif. To better understand LDN biosynthesis and regulation, we stably expressed the cDNA encoding the Caenorhabditis elegans ␤1,4-N-acetylgalactosaminyltransferase (GalNAcT), which generates LDN in vitro, in Chinese hamster ovary (CHO) Lec8 cells, to establish L8-GalNAcT CHO cells. The glycan structures from these cells were determined by mass spectrometry and linkage analysis. The L8-GalNAcT cell line produces complex-type N-glycans quantitatively bearing LDN structures on their antennae. Unexpectedly, most of these complex-type N-glycans contain novel "poly-LDN" structures consisting of repeating LDN motifs (-3GalNAc␤1-4Glc-NAc␤1-) n . These novel structures are in contrast to the well known poly-LN structures consisting of repeating LN motifs (-3Gal␤1-4GlcNAc␤1-) n . We also stably expressed human ␣1,3-fucosyltransferase IX in the L8-GalNAcT cells to establish a new cell line, L8-GalNAcT-FucT. These cells produce complex-type N-glycans with ␣1,3-fucosylated LDN (LDNF) GalNAc␤1-4(Fuc␣1-3)GlcNAc␤1-R as well as novel "poly-LDNF" structures (-3GalNAc␤1-4(Fuc␣ 1-3)GlcNAc␤1-) n . The ability of these cell lines to generate glycoprotein hormones with LDN-containing N-glycans was studied by expressing a recombinant form of the common ␣-subunit in L8-GalNAcT cells. The ␣-subunit Nglycans carried LDN structures, which were further modified by co-expression of the human GalNAc 4-sulfotransferase I, which generates SO 4 -4GalNAc␤1-4Glc-NAc-R. Thus, the generation of these stable mammalian cells will facilitate future studies on the biological activities and properties of LDN-related structures in glycoproteins.

Section: Discussionmentioning

confidence: 81%

Novel Poly-GalNAcβ1–4GlcNAc (LacdiNAc) and Fucosylated Poly-LacdiNAc N-Glycans from Mammalian Cells Expressing β1,4-N-Acetylgalactosaminyltransferase and α1,3-Fucosyltransferase

Kawar

Haslam

Morris³

et al. 2005

“…E-selectin appears to bind better to glycans containing PFPL than to short glycans (41)(42)(43). Thus, E-selectin might bind preferentially to the sialylated PFPL O-glycans of PSGL-1.…”

Section: Fig 5 Comparison Of Binding Of P-selectin Igg Chimera Tomentioning

confidence: 97%

Glycosulfopeptides with O-Glycans Containing Sialylated and Polyfucosylated Polylactosamine Bind with Low Affinity to P-selectin

Leppänen

Penttilä

Renkonen

et al. 2002

P-selectin glycoprotein ligand-1 (PSGL-1), a dimeric mucin on leukocytes, is the best characterized ligand for selectins. P-selectin binds stereospecifically to the extreme N terminus of PSGL-1, which contains three clustered tyrosine sulfates (TyrSO 3 ؊ ) adjacent to a Thr residue with a core 2-based O-glycan expressing sialyl Lewis x (C2-O-sLe x ). GSP-6, a synthetic glycosulfopeptide modeled after the N terminus of PSGL-1, containing three TyrSO 3 ؊ residues and a short, monofucosylated C2-OsLe x bound to P-selectin with high affinity (K d ϳ650 nM). However, PSGL-1 from human HL-60 cells contains higher levels of O-glycans that are sialylated and polyfucosylated polylactosamines (PFPL). Furthermore, studies with fucosyltransferase-deficient mice suggest that sialylated PFPL structures contribute to binding to P-selectin. To resolve whether sialylated PFPL O-glycans participate in binding of PSGL-1 to human P-selectin, we synthesized glycosulfopeptides, designated GSP-6 and GSP-6؆, with three TyrSO 3 ؊ residues and either difucosylated polylactosamine (C2-O-Le x -sLe x ) or trifucosylated polylactosamine (C2-O-Le x -Le x -sLe x ). Binding of the GSPs to P-selectin was measured by affinity chromatography, fluorescence solid-phase assays, and equilibrium gel filtration. Unexpectedly, both GSP-6 and GSP-6؆ bound to P-selectin with low affinity (K d ϳ37 M for GSP-6 and K d ϳ50 M for GSP-6؆). Binding of GSP-6 and GSP-6؆ to P-selectin required fucosylation and, to a lesser extent, sialylation as well as the sulfated peptide backbone of GSP-6 and GSP-6؆. These results demonstrate that contrary to expectations, a core 2 O-glycan containing sialylated PFPL does not promote high affinity binding of PSGL-1 to P-selectin.

“…Numerous reports have described the binding of E-selectin transfected cells to immobilized sLe xcarrying glycolipids (35,55,56), and it was also shown that E-selectin-transfected cells can roll on immobilized glycolipids under physiologic flow conditions (34). However, the contribution of cell surface-expressed glycolipids in the context of a cell surface glycocalyx to the binding of cells on immobilized Eselectin has not yet been analyzed.…”

Section: Binding Of Fuc-tiv and Fuc-tvii Transfectants To E-selectin-mentioning

confidence: 99%

The α(1,3)-Fucosyltransferase Fuc-TIV, but Not Fuc-TVII, Generates Sialyl Lewis X-like Epitopes Preferentially on Glycolipids

Huang

Laskowska²,

Vestweber

et al. 2002

Fuc-TIV and Fuc-TVII are the two ␣(1, 3)-fucosyltransferases in myeloid cells responsible for the biosynthesis of sialyl Lewis X (sLe x ), the minimal ligand structure for the selectins. We have compared the ability of Fuc-TIV and Fuc-TVII to generate sLe x -like epitopes in transfected Chinese hamster ovary (CHO)-Pro ؊ 5 cells expressing the P-selectin glycoprotein ligand-1 and the core-2 branching enzyme C2GnT. We found that mouse Fuc-TIV and Fuc-TVII can generate similar levels of cell surface sLe x . Surprisingly however, Fuc-TIV-generated sLe x was resistant to proteinase K and trypsin treatment and could be removed from cells by delipidation with chloroform/methanol, whereas 80 -90% of Fuc-TVIIgenerated sLe x was protease-sensitive, and most of it resistant to delipidation. Despite similar levels of sLe x on the cell surface, Fuc-TVII transfectants adhered to immobilized E-selectin-IgG under static and flow conditions better than Fuc-TIV transfectants. Binding was mainly protease sensitive, indicating that glycoproteins were more efficient ligands than glycolipids. In summary, we conclude that the two fucosyltransferases differ in their in vivo specificity for acceptor substrates with Fuc-TVII generating sLe x preferentially on glycoproteins, whereas most of the Fuc-TIV-generated sLe x is found on glycolipids. Interestingly, the non-catalytic portion of Fuc-TIV in a Fuc-TIV/VII chimeric enzyme mediated the specificity for glycolipid substrates.The three known selectins, L-, E-, and P-selectin are cell adhesion molecules that initiate interactions between leukocytes and endothelial cells during leukocyte extravasation (1). The minimal ligand structure for all three selectins is the tetrasaccharide sialyl Lewis X (sLe x ). 1